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Self-organization and symmetry breaking in intestinal organoid development

Nature volume 569, pages 66–72 (2019)Cite this article

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Abstract

Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.

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Fig. 1: Intestinal organoids development from LGR5+ and LGR5− single cells.
Fig. 2: Trajectory of organoid development.
Fig. 3: YAP1 target genes are transiently expressed during organoids development.
Fig. 4: Transient YAP1 activation is necessary for symmetry breaking.
Fig. 5: YAP1 cell-to-cell variability allows Notch–DLL1 activation and symmetry breaking.

Data availability

RNA-seq data are available at the Gene Expression Omnibus (GEO) under accession codes GSE115955 (bulk) and GSE115956 (single cells).

Code availability

Code used for image analysis in this study was developed in the Liberali laboratory in Matlab and Python 3. For Python 3, multiple open source Python libraries for scientific computing and image analysis were used. The code for organoid 2D and 3D segmentation, feature extraction and organoid linkage is available under https://github.com/fmi-basel/glib-nature2018-materials. An R implementation of the Griph algorithm for scRNA-seq analysis is available as an R package at https://github.com/ppapasaikas/Griph.

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Acknowledgements

We thank F. Maurer for organoid preparation, E. Tagliavini for IT support, G. de Medeiros for help in light-sheet imaging, A. Peters for light-sheet, L. Gelman for assistance and training, H. Kohler for sorting, S. Smallwood and S. Thiry for sequencing, R. Cuttat and S. Schuierer for scRNA-seq analysis, L. Pelkmans, J. Betschinger, D. Gilmour, L. Giorgetti, C. Tsiairis and laboratory members for reading the manuscript. Funding: SNSF (POOP3_157531 to P.L.). This work received funding from the ERC under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 758617).

Reviewer information

Nature thanks Anne Rios and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. Andrea Boni & Petr Strnad

    Present address: Viventis Microscopy Sàrl, EPFL Innovation Park, Lausanne, Switzerland

  2. These authors contributed equally: Denise Serra, Urs Mayr, Andrea Boni

Authors and Affiliations

  1. Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland

    Denise Serra, Urs Mayr, Andrea Boni, Ilya Lukonin, Markus Rempfler, Ludivine Challet Meylan, Michael B. Stadler, Petr Strnad, Panagiotis Papasaikas, Dario Vischi & Prisca Liberali

  2. University of Basel, Basel, Switzerland

    Denise Serra, Urs Mayr, Ilya Lukonin & Prisca Liberali

  3. Swiss Institute of Bioinformatics, Basel, Switzerland

    Michael B. Stadler & Panagiotis Papasaikas

  4. Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland

    Annick Waldt & Guglielmo Roma

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Contributions

P.L. conceived and supervised the study, P.L., D.S., U.M. and A.B. designed the experiments, D.S., U.M., L.C.M. and A.B. performed the time courses, I.L. performed the first YAP1 experiments, U.M. and A.B. designed the image analysis, M.R. and D.V. developed 3D and single-cell imaging, U.M. developed the trajectory inference, A.B. and P.S. built the light-sheet, A.B. and U.M. performed the light-sheet experiments, A.B. performed light-sheet image analysis, D.S. performed all transcriptomics experiments, M.B.S. designed transcription factor motifs analysis, D.S. and M.B.S. performed the analysis of RNA-seq data, M.B.S. and P.P. developed Griph, A.W. and G.R. performed the scRNA-seq analysis, U.M. performed the multiplexing experiments, P.L., D.S., U.M. and A.B. wrote the paper.

Corresponding author

Correspondence to Prisca Liberali.

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Competing interests

A.B. and P.S. are co-founders of Viventis Microscopy Sàrl that commercializes the light-sheet microscope used in this study.

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Extended data figures and tables

Extended Data Fig. 1 Intestinal development of LGR5+ and LGR5− organoids.

a, LGR5+ and LGR5− single cells sorted from LGR5::DTR-eGFP reporter mouse 3 h after FACS (left), with box plot showing LGR5::DTR-eGFP intensity quantification (right) (n = 602 organoids for LGR5+; n = 576 organoids for LGR5−). Box plot elements show quartiles, and whiskers denote 1.5× the interquartile range. b, FACS histogram of GFP signal of LGR5::DTR-eGFP single cells. Dashed boxes depict gating. c, Representative images of 4i imaging showing the same organoids stained with DAPI, GFP, lysozyme, proliferating cell nuclear antigen (PCNA), aldolaseB and Cell Trace. d, Distribution of eccentricity at 120 h of development for LGR5+ and LGR5− starting populations (LGR5+ n = 463 organoids; LGR5− n = 711 organoids). e, Representative images of LGR5+ and LGR5− cells at 120 h of development. Bottom, enlargements depict budding organoids and enterocysts. f, Representative images of enterocysts stained with DAPI, different cell-type and proliferation markers. g, Left, representative images of organoids grown from LGR5+ and LGR5− single cells in the presence of Wnt signalling inhibition (IWP-2, 2 µM) or activation (CHIR99021, 5 µM). Organoids are fixed at 120 h and stained for nuclear marker (DAPI) and enterocyte marker (aldolaseB). Top row shows complete well overview; bottom row shows magnified examples of single organoid. Right, quantification of enterocysts at 120 h of organoid development after Wnt signalling inhibition (IWP-2, 2 µM) or activation (CHIR99021, 2 µM) (n = 2 replicates). Data are mean values.

Source Data

Extended Data Fig. 2 Feature selection for organoid development.

a, Pearson correlation heat map of the 14 selected features used for trajectory inference (n = 23,421 organoids). b, Fourteen selected features are grouped based on the underlying information. c, Comparison between eccentricities calculated on projected images and on 3D volumes (n = 16,175 organoids). d, Number of in-focus planes at different time points in cleared and uncleared organoids (n = 2,310 organoids). Planes are classified as in-focus with a logistic regression using the ratio of maximum in-plane intensity to maximum intensity in the whole stack as feature. Shading denotes s.d. e, Diffusion maps for pooled LGR5+ and LGR5− organoids (14 selected features, n = 23,421 organoids). Colour-coding denotes starting population and pseudotime. f, Diffusion maps for LGR5− (first column, 14 selected features, n = 13,623) and LGR5+ (second column, 14 selected features, n = 9,798) organoids, and for the pooled dataset with the full feature set (third column, 66 features, n = 23,421). Colour-coding denotes enterocytes (aldolaseB), PCNA, Paneth cells (lysozyme), organoid area, eccentricity and enterocyst classification (in which class 1 denotes enterocysts).

Source Data

Extended Data Fig. 3 Cell types and proliferation markers along organoid development trajectories.

Proliferation (PCNA) and cell-type marker (lysozyme and aldolaseB) trends on pseudotime for LGR5+ (n = 9,798) and LGR5− (n = 13,623) organoids.

Source Data

Extended Data Fig. 4 Dual illumination inverted light-sheet imaging of organoids.

a, Cross-sectional view of objective mounting block showing illumination paths (light blue dashed line) and imaging path (green dashed line). Illumination and imaging objective are solid mounted in the aluminium block and immersed in water contained in the reservoir (black arrow). b, Side view of microscope objectives arrangement: the sample embedded in Matrigel (red arrow) is held on the FEP membrane (black arrow) mounted in a sample holder and positioned between the two illumination objectives. c, Organoids handling procedure: grown organoids are dissociated and single cells are embedded in Matrigel. Several Matrigel drops can be transferred into the imaging chamber allowing imaging parallelization. d, Illustration of image improvement by double illumination. Scattering of single illumination beam by the organoid prevents complete and homogenous illumination (red arrows). This problem is minimized by double illumination. Scale bar, 25 µm. e, Representative images of an organoid expressing H2b–mCherry (red) and membrane-bound GFP (green). Slices every 20 µm across the organoid volume are shown. Scale bar, 25 µm. f, Workflow of light-sheet data analysis. g, Morphological features (major axis, area and volume) derived from light-sheet imaging. Budding organoids n = 6, enterocysts n = 3. h, Dynamic time-warp mapping of light-sheet data onto the trajectory. Budding organoid branch for mean area progression inferred from the trajectory (orange line, n = 23,421) and mean area progression extracted from time-lapse light-sheet imaging (violet line, n = 6) before (left) and after (right) morphing. Red dots indicate positions of real time (h) relative to pseudotime. Shading denotes s.d.

Source Data

Extended Data Fig. 5 LGR5 dynamics during organoid development.

a, Representative images of organoids grown from LGR5+ and LGR5− starting populations at 0, 24 and 120 h of development. b, Cell types and proliferation markers plotted on pseudotime for LGR5+ (top, n = 9,798) and LGR5− (bottom, n = 13,623) starting populations. c, Light-sheet time-lapse imaging of organoid formation starting from a single LGR5+ cell (GFP signal) expressing membrane-bound mCherry (MEM9–mCherry). Green arrows are pointing to cells re-acquiring GFP signal from LGR5 reporter in the organoid crypts. d, Quantification of GFP signal of LGR5 reporter from time lapse shown in c. Cells localized in the organoid crypt are plotted in green, cells localized outside the crypt and on the main body are plotted in grey (n = 91 cells).

Source Data

Extended Data Fig. 6 Bulk RNA-seq analysis.

a, Principal component analysis (PCA) analysis of time-course (0–120 h) bulk RNA-seq shows no mouse or batch effect (n = 3 biological replicates). b, Unsmoothed gene expression profile mapped on pseudotime trajectory of stem cell marker Olfm4. c, Unsmoothed gene expression profiles mapped on pseudotime trajectory of cell-type-specific genes. Paneth cell: Lyz1; ISC: Lgr5; enterocyte: Apoc3; goblet cell: Muc2; enteroendocrine cell: Sst. d, Left, protein abundance and gene expression profile of ISC marker Lgr5 mapped on pseudotime trajectory. Middle, protein abundance and gene expression profile of Paneth cell marker Lyz1 mapped on pseudotime trajectory. Right, protein abundance of enterocyte marker aldolaseB and gene expression profile of enterocyte marker Apoc3 mapped on pseudotime trajectory. e, Smoothed and unsmoothed gene expression profile of canonical Wnt target gene Axin2, Fzd2 and non-canonical target genes Nfatc2 and Rac1. f, Mean expression profile for each cluster and relative quantiles (0.05, 0.25, 0.50, 0.75 and 0.95). Annotation enrichments are shown on the right. E.S., enrichment score. g, Unsmoothed gene expression profile of Yap1, Tead4 and Ggta1 and top three transcription factors (Tead1, Tead4 and Fosl1) contributing to differential gene expression between 24 h and 0 h. Data in b, c, e, g are mean and s.d. (n = 3 biological replicates).

Source Data

Extended Data Fig. 7 YAP1 is necessary but not sufficient for organoid formation.

a, Fraction of cells per organoid having YAP1 in the ON state (green, nuclear localization) or in the OFF state (red, cytoplasm localization) over time (n = 1,074 organoids). b, Entropy of YAP1 states (that is, variability in YAP1 activation in ON and OFF states) over time (n = 1,074 organoids). Shading denotes s.d. c, Quantification of YAP1 mean intensity after Wnt removal at 48 h or 72 h in organoids fixed at 72 h or 96 h (n = 4 replicates). Violin plot lines show quartiles. d, Images of organoids derived from single cells isolated from organoids at 72 h or at 120 h. e, Organoids treated at different time points with control or with verteporfin. Fixation at 96 h. Left, representative images; right, efficiency quantification (normalization: control, n = 2 replicates). Bar plots depict the mean. f, YAP1 overexpressing organoids with or without Wnt. Fixation at 96 h. Left, representative images; right, efficiency quantification (normalization: control, n = 4 replicates). P = 0.001, two-sided t-test. g, Left, images of organoids with Wnt removal at 48 h, or after double knockout of LATS1 and LATS2 (LATS DKO). Top, fixation at 72 h. Bottom, fixation at 96 h (Wnt removal at 48 h) or 120 h (LATS DKO). Right, quantification of enterocysts as retrieved in the left panel (n = 4 replicates for Wnt removal at 48 h; n = 3 replicates for LATS DKO). P = 0.0001 (Wnt removal); P = 0.002 (LATS DKO), two-sided t-test. h, Representative images of control, verteporfin treatment at 48 h, Wnt removal at 48 h, YAP1 overexpression, LATS DKO or treatment with EREG at 0 h. Top, fixation at 72 h; bottom, fixation at 96 h (Wnt removal at 48 h, YAP1 overexpression, LATS DKO) or 120 h (control, verteporfin 48 h, EREG). i, Scatter plot of Flag mean intensity signal and YAP1 mean intensity signal (left) and of Flag mean intensity and aldolaseB mean intensity (right) in YAP1-overexpressing organoids at 96 h. Data are mean ± s.d.

Source Data

Extended Data Fig. 8 Griph robustness analysis and comparison to other methods.

Comparison of Griph lower dimensional embedding (Griph/LargeVis) to different embedding approaches (PCA, PCA combined with distributed stochastic neighbor embedding (t-SNE), diffusion maps) and analysis of method sensitivity for variable gene selection. Five different sets of variable genes have been selected (using Griph to retain 10%, 25% or 50% of genes per bin, by Michaelis–Menten fitting of the gene dropout rates as implemented in M3Drop, or by the previously described mean-variance fitting procedure48) and analysed. a, b, First and second dimension are shown and results are colour-coded for enterocyte marker genes (as in Extended Data Fig. 9b) (a) and YAP1 target genes (as in Fig. 5b) (b). n = 1,863 cells.

Extended Data Fig. 9 scRNA analysis.

a, Experimental workflow for scRNA-seq. b, Griph-based visualization of single-cell degree of expression of marker genes for stem cells, Paneth cells and enterocytes. c, Griph-based visualization of single-cell degree of expression of marker genes for transient-amplifying cells, goblet cells, enteroendocrine cells, tuft cells, immature proximal enterocytes, immature distal enterocytes, transient-amplifying cells G1-phase, transient amplifying cells G2-phase, early progenitor enterocytes, late progenitor enterocytes, mature proximal enterocytes, mature distal enterocytes, enterocytes (villus bottom), enterocytes (villus middle), enterocytes (villus top) and enterocytes. d, Spearman correlation between expression of YAP1 target genes and expression of Dll1, Dll4, Jag1, Jag2 and Atoh1 in single cells at 120 h not expressing Paneth, goblet, enteroendocrine, enterocyte and stem-cell markers (n = 696 cells).

Source Data

Extended Data Fig. 10 YAP1 cell-to-cell variability allows a Notch–DLL1 event and symmetry breaking.

a, Images showing YAP1 variability and DLL1+ cells. Magnified view denotes nuclear localization of HES1 in the same organoid. Arrows: red (DLL1+ cells), yellow (HES1+ cells). Scale bar, 10 µm. b, Images showing overlap of Paneth cells with DLL1+ cells. c, MIP images (top) and magnified single plane images (bottom) of: control, verteporfin addition at 48 h, Wnt removal at 48 h, LATS double-knockout (DKO) and EREG treatment. Organoids are fixed at 72 h. d, MIP images (top) and magnified single plane images (bottom) of: control, verteporfin addition at 48 h, Wnt removal at 48 h, YAP1 overexpression, LATS DKO and EREG treatment. Organoids are fixed at 72 h. e, Fraction of enterocysts (left) and fraction of organoids with Paneth cells (right) for control and organoids treated with Ly411575 or MK-0752 at 0 h. Fixation at 120 h (normalization: control, n = 4 replicates). Ly411575 P = 0.009; MK-0752 P = 0.003, two-sided t-test. Data are mean ± s.d. f, MIP images (top) and magnified single plane images (bottom) of organoids treated with control or DAPT at 120 h and fixed at 144 h and stained for AldoB (left) or LYZ (right) g, Top, annotation enrichment of genes correlated with expression of YAP1 target genes in cells expressing YAP1 target genes and Dll1, Dll4, Jag1, Jag2 and Atoh1 genes. Bottom, annotation enrichment of genes anti-correlated with YAP1 target genes expression in cells expressing YAP1 target genes and Dll1, Dll4, Jag1, Jag2 and Atoh1 genes. E.S., enrichment score. h, MIP images of organoids at 120 h, showing the canonical Wnt signalling response (TCF–GFP) in cells neighbouring Paneth cells.

Source Data

Supplementary information

Supplementary Information

This file contains Supplementary Methods and additional references.

Reporting Summary

Supplementary Table 1

Bulk and single cell RNA sequencing analysis. Bulk RNA sequencing time course: Rpkm replicates, normalized mean expression, differential expression 24h-0h (n=3 biological replicates for each time point, statistical test used: Genewise Negative Binomial Generalized Linear Models With Quasi-Likelihood Tests, factors considered: time point, mouse and Dnase treatment) and Beta-coefficient for transcription factor binding motifs analysis. Single cell RNA sequencing: list of Yap1 target genes and intestinal epithelium cell types specific genes used within this study.

Supplementary Table 2

A list of primary and secondary antibodies used within this study.

Video 1: Time lapse Video of a budding organoid expressing H2b-mCherry as nuclear marker

A full stack is acquired every 10 minutes for around 5 days. Left panel –3D volume rendering shows that the whole organoid volume is imaged. Right panel – single plane intersecting the middle of the organoid volume. Experiments were repeated at least three times.

Video 2: Time lapse Video of an enterocyst expressing H2b-mCherry as nuclear marker (red) and membrane bound GFP (mem9-GFP, in green) imaged by light-sheet microscopy.

A full stack is acquired every 10 minutes for around 5 days. Left panel –3D volume rendering shows that the whole organoid volume is imaged. Right panel – single plane intersecting the middle of the organoid volume. Experiments were repeated at least three times.

Video 3: Left panel - 3D volume rendering of segmented light sheet data for one budding organoids and one enterocyst. Right panel – Dynamic plots of budding organoid and enterocyst volume and major axis over time.

Experiments were repeated at least three times.

Video 4: Time lapse Video of a budding organoid expressing Lgr5::GFP reporter (green) and membrane bound mCherry (mem9-mCherry, red) imaged by light-sheet microscopy.

A full stack is acquired every 30 minutes for around 5 days. Left panel –3D volume rendering shows that the whole organoid volume is imaged. Right panel – single plane intersecting the middle of the organoid volume. At the end of time lapse a slide through the all volume is shown where the presence of Lgr5+ cells in the crypt is visible. Experiments were repeated at least twice.

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Serra, D., Mayr, U., Boni, A. et al. Self-organization and symmetry breaking in intestinal organoid development. Nature 569, 66–72 (2019). https://doi.org/10.1038/s41586-019-1146-y

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